Abstract The public health problem caused by opiate abuse in the US has had consequences for pregnant women and their babies. The incidence of the neonatal abstinence syndrome (NAS), which is a constellation of narcotic drug withdrawal symptoms that develops in infants born to narcotic dependent mothers, has risen dramatically. Treatment of infants with NAS often requires a prolonged hospitalization and lengthy administration of opioid containing medications, which can affect infant development. Despite its emergence as a very costly public health problem, there are no preventative treatments for NAS. Previous and current NAS research programs generally explore alterations in the dose or type of narcotic drug used to treat an infant with established NAS. The first aim of this study is to complete a clinical trial that tests a novel approach for preventing NAS. Based upon a genetic discovery, we demonstrated that administration of a 5-HT3 antagonist (ondansetron) prevented the symptoms of narcotic drug withdrawal in experimental studies in mice and in adult humans. From this, we hypothesize that ondansetron administration to pregnant mothers with opiate use disorder (OUD) just prior to delivery, followed by a brief period of ondansetron administration to the neonate, could reduce the incidence or severity of NAS. Ondansetron is a broadly utilized drug with a substantial safety record in pregnant women and infants. Therefore, we will complete a multi-center, randomized, double blind, and placebo-controlled trial to determine whether ondansetron treatment will reduce the incidence (percentage of infants requiring pharmacotherapy for NAS) or severity (total amount of opiate drug administered, length of hospitalization, NAS symptom severity scores) of NAS in babies born to mothers with OUD. This is the only study that tests a novel and generally innocuous treatment that will prevent NAS. If a brief period of ondansetron administration can prevent the development of significant NAS, this would provide a desperately needed preventative therapy, which would reduce human suffering and the growing societal costs associated with opiate abuse. The second aim of this study is to develop an innovative new method [Capillary microsampling, (CMS)], which uses a very small volume of blood (8 ul) to accurately measure drug levels in neonates. We have modified this method to enable its use in a neonatal care setting. The results obtained using the optimized CMS method (iso-CMS) will be validated in a clinical setting by determining whether iso-CMS results accurately reflect simultaneously measured plasma ondansetron concentrations in adult human subjects. We will then use iso- CMS to measure ondansetron levels in study neonates. We will also demonstrate that that iso-CMS can reliably measure the concentrations of at least 5 other drugs. This innovative new method will enable the dosing regimens for drugs with a narrow therapeutic index to be adjusted in neonates.